Project Summary/Abstract Connectomics is a new field, created with the goal of densely or completely mapping the connections in the brain. Because this goal is at present only achievable for small organisms, connectomics has taken on two forms in the study of larger brains. Macroscale connectomics is used to describe the connections between brain areas, which in experimental animals is achieved with tracers, while humans it is typically pursued at a very coarse scale with diffusion imaging, a form of MRI. Microscale connectomics aims to create dense wiring diagrams of local circuits, small volumes of the brain with neurons that form rich networks within a local neighborhood. Macroscale connectomics lacks cellular resolution, while microscale connectomics can only reconstruct small volumes, with little information about the source of inputs entering the volume, or the targets of axons exiting it. We propose to develop a technique to bridge the gap between the microscale and the macroscale by creating experimental and analytical methods for mapping individual axons over long distances, concentrating on the largest 50% of myelinated axons (long-distance ?wires?) in the brain. For the current RFA to develop ?Tools to Facilitate High-Throughput Microconnectivity Analysis? we are specifically targeting one of the recommended goals, to ?Develop a high-quality toolbox of methods for efficiently mapping and annotating projections? in the human brain. The method?based on high-resolution 3D imaging of antibody-stained axons?can be scaled to analyze entire brains, but here we propose to apply it to the posterior pole of a macaque brain, a large (~10 cm3) volume will contain >20 visual cortical areas. The goal is to trace most of the larger axons (>1 um) between these areas, thus creating a dense axonal connectomics data set from the cortical visual system, allowing us to examine not only the hierarchy of visual areas, but also the structure of computational maps. Ultimately, this approach will permit whole-brain analysis of axonal projections, with targeted examination of key circuits for microscale connectomics. While microscale connectomics of whole human brains will remain impossible for the foreseeable future, dense axonal mapping should be achievable in the next 5 to 10 years. This proposal offers a pathway towards this new type brain-wide anatomy: Axonal Connectomics.